The development of a coupled in vitro protein synthesizing system of B. subtilis extracts is proposed. This system will be used to study the regulation of the expression of the genes for alpha-amylase, dCMP deaminase, maltase and acid soluble spore proteins (alpha beta). In the development of this in vitro system we propose to ascertain the fidelity of the translational portion of the system by synthesizing specific enzymes in vitro. We have succeeded in making enzymatically active dCMP deaminase using phage SP01-induced mRNA as the template in our in vitro system. In addition the spore proteins alpha beta have been synthesized in vitro. The amount of an individual protein synthesized in vitro is in the nanogram range with as much as 200 nanograms of alpha beta protein being synthesized. We are developing assays for alpha-amylase and maltase that will be sufficiently sensitive to detect nanogram quantities of these enzymes. A radioimmune assay for alpha-amylase is being developed to complement an enzymatic assay which has sensitivity to the desired range. For use as templates in the in vitro system the genes for the proteins mentioned above are being cloned. Ultimately the in vitro system will be used to assess the role(s) of changes to RNA polymerase, unusual nucleotides, etc. on gene expression.